CELL-FUSION TO STUDY NUCLEAR-CYTOPLASMIC INTERACTIONS IN ENDOTHELIAL-CELL APOPTOSIS

Studies examining the regulation of nuclear rearrangements during apop tosis have led to conflicting results. Cytoplasmic control of nuclear events has been strongly suggested by cell-free experimental systems. In contrast, strict cytoplasmic control cannot account for the results of fibroblast-thymocyte fusion experiments in which dexamethasone ind uction of polykaryons led only to thymocyte nuclear apoptosis. Unresol ved by these fusion studies was whether fibroblast nuclei were indiffe rent to heterologous cytoplasmic signals. Our objective was to resolve this discrepancy using cell fusion in a homologous system. Our strate gy was to fuse endothelial cells with high levels of susceptibility to the induction of apoptosis (log phase cells arrested in G(1) for 48 h ours by isoleucine deprivation) with those manifesting low levels of s usceptibility (serum-deprived, G(0)). Resultant fused and unfused cell s were induced to undergo apoptosis by incubation with tumor necrosis factor-alpha and cycloheximide. Depending on the parental cell of orig in, between 14 and 30% of dikaryous contained one apoptotic and one in tact nucleus, indicating that strict cytoplasmic control was not occur ring. In accord with this, the total frequency of nuclear apoptosis wa s unchanged after fusion. However, the distribution of apoptotic nucle i revealed a pronounced cytoplasmic influence, with a two- to fivefold increase in coordinate nuclear behavior. This pattern of nuclear apop tosis was consistent with a model of control in which both the state o f nuclear susceptibility to apoptosis and expression of cytoplasmic pr o-apoptotic regulators determined whether nuclear apoptosis would even tuate.